*4.1.3 Treatment with supercritical CO2*

Supercritical fluid is a special state of matter, which exists at elevated temperature and pressure above its critical point and beyond the distinct liquid and gas phases. For carbon dioxide (CO2), this supercritical phase can be reached by subjecting it to pressures over 73 bar and temperatures over 31°C (**Figure 8**). Under these conditions, CO2 is neither liquid nor gaseous, but combines the properties of both states. Its "gaseous"

*State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

properties give CO2 a very high diffusion capacity through a treated material (e.g., cork), while its "liquid" behavior provides a very high extraction power toward some volatile molecules (e.g., volatile and malodorous compounds of cork, including TCA). By adjusting the pressure/temperature conditions (e.g., 120 bars/60°C), it is possible to optimize the extraction of TCA from cork by CO2 while preserving the mechanical

**Figure 8.** *Pressure–temperature phase diagram for CO2.*

#### **Figure 9.**

*Supercritical CO2 cleaning technology (Diamant®) to remove TCA and other volatile compounds from cork granules.*

properties of the cork material [92]. This extraction process does not require the use of organic solvents, which makes it safe for human health and environmentally friendly.

Diamant® (Diam Bouchage) was the first industrial system for supercritical CO2 cleaning of cork material based on a technology patented over 20 years ago (**Figure 9**) [93]. It has been shown that this cleaning process is highly effective in achieving "zero" levels of residual TCA (i.e., below LOQ = 0.3 ng/L) in a single treatment cycle of cork granules, which had an initial contamination close to 20 ng/L of *releasable TCA* [92], and later close to 50 ng/L [94]. In addition, over 150 other molecules besides TCA are also removed (mainly nonpolar), including various terpenes, pyrazines, etc. [95, 96]. Further development of supercritical CO2 extraction technology for cork material involved optimization of the used energy and the CO2 volume. A recent example of other technologies based on the similar principle is Xpür® (Amorim Cork), which was also designed to clean cork granules.

Generally, the existing supercritical CO2 extraction technologies for cork material are limited to cork granules, which are subsequently used to produce agglomerated stoppers. Applying this process to natural cork stoppers encountered certain difficulties. The process efficacy was greatly reduced due to the low diffusion of supercritical CO2 in the cork structure: when growing on a tree, the cork acquires a nonisotropic internal structure, i.e., its physical and mechanical properties (elasticity) are not the same and depend on the orientation of the cork growth lines. During the supercritical CO2 cleaning process involving pressurization and decompression, the cork compresses and then decompresses unevenly, generating fractures in the material. This results in delamination of cork growth veins, a loss of its physical properties of about 30%, and a significantly increased heterogeneity of oxygen permeability levels among cleaned natural cork stoppers. In turn, microagglomerated cork stoppers made of cork granules provide far superior homogeneity and consistency.

Supercritical CO2 extraction was proposed also for the determination of *total TCA* in ground corks [69]. In addition, this technology is widely used nowadays in other industries, as it allows the treatment of raw materials at moderate temperatures avoiding side processes (e.g., Maillard reactions) and the formation of undesirable by-products. Thus, it is commonly used in perfumery to extract aromatic molecules from natural materials, in the food industry to extract caffeine from coffee (producing decaffeinated coffee), theine from tea, lupulin from hops, etc.

#### **4.2 Quality control techniques: selection of "TCA-free" cork stoppers**

As it was mentioned in Section 3, the analysis of *releasable TCA* is used for the quality control of cork batches. The corks are randomly selected, analyzed, and the results are extrapolated to the entire batch of stoppers. Therefore, a purchaser of these natural corks can count on the probability of contamination within the batch, but not on the specific TCA contamination of each individual cork. To guarantee the "TCA-free" status of each stopper, they need to be analyzed individually, one by one. The usual *releasable TCA* method is not suitable for this goal and is considered destructive: soaking and following drying procedures alter the cork surface due to tannin staining [97] and other effects. Therefore, the aim was to develop nondestructive methods, which could correlate with the *releasable TCA* analysis. As a result, "TCA-free" corks can be selected from the analyzed batch, commercialized, and used later for wine bottling. Nowadays, there are two main nondestructive approaches to the individual cork analysis, which will be discussed below: sensory methods and automated methods.

*State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

#### *4.2.1 Sensory methods*

The high interest in the sensory evaluation of corks in the late 1980s and 1990s led to the development of the first protocols for analysis of stoppers [98, 99]. In 1996, a typical sensory method of cork analysis was elaborated at the Hochschule Geisenheim University (former Forschungsanstalt Geisenheim), which according to the latest issue [100] offers the following procedure:


Other routine sensory evaluation methods of cork stoppers can vary somewhat in terms of flask volume, amount of water added, etc. [97, 101]. For example, Macku and colleagues [97] used 125 mL flasks with six drops of water. At the same time, the principles described above remain the same and are often referred to as "dry soak" sensory screening methods. The advantage of the sensory method also lies in the possibility to identify various aroma deviations related not only to TCA and haloanisoles. Among other off-odor compounds are geosmin, 2-methoxy-3,5-dimethylpyrazine, and various malodorous molecules, including those formed due to improper treatment of cork material during the production process.

To prove the effectiveness of the "dry soak" method, Macku and colleagues [97] performed an extensive sensory evaluation of 2000 corks. As a result, about 6% of the stoppers were rejected and then analyzed by GC–MS. About one-third of the rejected corks possessed *releasable TCA* levels above 1 ng/L, while the rest had levels below 1 ng/L (their discard can be related to the presence of other taint substances in cork). In turn, 100 stoppers from the "clean" group were randomly selected and also analyzed by GC–MS. None of these stoppers demonstrated a *releasable TCA* level higher than 1 ng/l, which is usually under the human perception threshold.

The "dry soak" method can be used for sensory screening of corks on an industrial scale. For example, the company Cork Supply adopted this technique for their natural corks, and selected "cork taint-free" stoppers became available to customers. Despite the proven effectiveness of the method, it is a time-consuming technique based on human factors, which can only be applied to a limited number of corks over a given period of time. Therefore, the market was waiting for automated methods of cork stoppers selection.

#### *4.2.2 Automated methods*

The purpose of automated methods is to quickly analyze each individual cork stopper for TCA content and then separate the corks into different groups depending on the TCA contamination. The general technical principle for cork analysis is as follows: a cork stopper is placed into a small hermetic chamber and heated, which induces vaporization of TCA from the cork; then the air from the chamber is collected and analyzed by GC–MS method with various detection systems [electron capture detector (ECD), ion mobility spectrometry (IMS), etc.].

Several companies have recently been developing such automated nondestructive technologies for the analysis of individual corks. The first system based on this principle, which started to work on an industrial scale, was NDTech® (Amorim Cork). Optimization of the technology allowed reduction of the time of analysis of one cork

to 15 seconds and provide the *releasable TCA* detection level of 0.5 ng/L. Thus, all analyzed cork stoppers with TCA levels below 0.5 ng/L are selected as "TCA-free" corks. Among other automated systems present on the market or in the commercial phase are the following: the system of CEVAQOE laboratory; Vocus Cork Analyzer (Tofwerk); the system of Cork Supply Portugal, S. A. (cork company); the system developed in collaboration between Bruker (scientific instruments manufacturer) and Egitron.

Automated systems for the analysis of TCA in corks are more efficient than sensory methods. However, considering the cork market, which requires billions of stoppers per year, even the automated methods available cannot analyze all the corks produced. Therefore, these technologies remain focused rather on higherquality corks for wines in the medium- and high-price segments.

## **5. Removal of TCA from contaminated wines**

Approaches to remove TCA from contaminated wines have been developed over several decades. Haloanisoles are nonpolar compounds; therefore, various hydrophobic materials (including different polymers) have been tested as candidates for diminishing TCA content in tainted wines (**Table 3**). Polyethylene, as a widespread and inexpensive plastic material, has shown high scalping properties in relation to TCA. It has been used in the form of a film [46] or granules (ultrahigh-molecular-weight polyethylene (UHMW PE). In general, polyethylene is able to absorb more than 90% of TCA [46, 102] and other haloanisoles from wine [46]. The efficiency of immersed film treatment depended on the film thickness, contact surface, and contact time. In the case of granules, tainted wine can be passed through the polymer particles, and the optimal rate should be applied. Other plastic items such as wine cask bladders and polypropylene lids also have scalping effects on haloanisoles [46]. A limitation for the use of plastic materials to reduce the TCA content in wine is related to the simultaneous scalping of wine color and aroma compounds [102], which can lead, in particular, to the loss of floral/fruity aromas [46]. In a recent study, the application of alimentary film (confidential composition) reduced TCA content by 81–83% after 48 h of wine-film contact [103]. Checking other wine components after this treatment showed no noticeable impact either on the color of red wines or on the phenolic and tannin composition. As for wine aroma compounds, there was no effect on the woody aroma profile; however, long-chain ethyl esters (ethyl octanoate, ethyl decanoate, and ethyl dodecanoate) were significantly absorbed, by about 70–80% after 48 h. Similar effects were also observed for synthetic bottle stoppers, which demonstrated higher absorption of the mentioned ethyl esters compared with corks [112].

Cork material itself can serve as a good absorbent of TCA and other halonisoles. It was found that cork stoppers are able to reduce the TCA content in tainted bottled wine by about 50% after 3 months of storage [46]. These results were similar for corks of different qualities, including agglomerated stoppers. Obviously, in order to reduce the TCA content in wine, corks should not be initially contaminated with TCA. Immersion of cork stoppers in tainted wine (soaking) can remove even more TCA, about 80–90% [46]. This idea has already been discussed in the previous section about the analysis of *releasable TCA*.

Subsequent works on the development of suitable polymeric materials for the removal of TCA from wine involved the usage of polyaniline-based materials and cross-linked derivatives of polyamidoamine [104]. They demonstrated a relatively high TCA absorption (>75%) and almost no impact on phenolic compounds in wine. At the same time, more research is required on the scalping of aroma compounds by these polymers. In order to eliminate tainted compounds selectively, the application of molecularly imprinted polymers (MIPs) was proposed. Tests with absorbents

*State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*


#### **Table 3.**

*Methods and materials proposed for the treatment of TCA-contaminated wines.*

of this type allowed the removal of TCA with a very high efficiency, >99% [105]. Simultaneously, it also revealed high retention properties toward other molecules such as 4-ethylphenol, 4-ethylguaiacol, oak lactones, 2-phenylethyl acetate, etc. Therefore, succeeding research on the absorption of other wine aroma compounds is also needed.

Among the inorganic materials, it was initially proposed to use activated charcoal. It demonstrated good results in TCA retention, but also low selectivity, i.e., high absorption of other wine components. Therefore, only slightly tainted wines are recommended to be treated with activated charcoal at doses, which are well below the maximum allowed levels (100 g/hL) in the EU for wine production [27, 113].

In this regard, zeolites, aluminosilicate minerals, seem to be more suitable absorbents. Zeolites possess a microporous structure, represented by a complex system of cavities (< 2 nm) and channels with a negatively charged surface. Due to these particularities, zeolites, as molecular sieves, have a good potential to interact and retain various molecules, including TCA. Zeolite powder can be directly mixed with contaminated wine [106] or integrated into filter plates that facilitates its industrial application. It was demonstrated that filtration of contaminated wines (5–20 ng/L of TCA) through such filters ("Fibrafix® TX-R") diminishes the TCA content to 1.1–1.2 ng/L (**Figure 10**), which is usually below the sensory thresholds [108]. In turn, in the wines contaminated with TBA (5–20 ng/L), undetectable levels of the pollutant were found after the treatment. Filtration through "Fibrafix® TX-R" plates had no significant impact on the analyzed wine aroma compounds (mainly secondary, fermentation aromas). At the same time, sensory panelists were able to distinguish between the wines filtered through the zeolite filter and a conventional filter, but no preference was given to any of the wines. As for the migration of aluminum ions from the filter sheet into the wine, it was insignificant, maximum 0.4 mg/L [108]. The application of zeolite containing filters is also described in the International Oenological Codex of OIV [109], and the recent EU Regulation (2019/934) permits the wine treatment using filter sheets with Zeolites-Y (Faujasite) for the selective removal of haloanisoles [107].

One of the gentle methods of TCA absorption involves the wine treatment with yeast hulls [110]. Several doses of yeast hulls were tested: from 100 mg to 800 mg per 1 L of wine. The effect of such treatment was moderate for TCA: the average dose (400 mg/L) provided only a limited reduction of TCA by 27%. As for other haloanisoles, they were absorbed in larger amounts: 55% for TeCA and 73% for PCA. Wine color deviation was measured for the treated wines and was minor even at the maximal dose of yeast hulls: decrease of color intensity by 3.1% (sum of OD at 420, 520, and 620 nm). Further studies about the impact of yeast hulls on the wine aroma composition can be of interest.

Among biogenic products that have also been tested to diminish TCA in wine are grape seed oil and milk products [111]. The latter exhibited a limited reduction of TCA content in wine, while the treatment with grape seed oil provided even better TCA scalping properties than plastic film. This fact demonstrates the potential of various natural products as absorbents, but the sensory effect on the wine of the used products was noticeable during tastings. The practicality, costs, and compositional consistency of these biogenic absorbents should also be taken into

**Figure 10.** *Removal of TCA by wine filtration through "Fibrafix® TX-R" [108].*

#### *State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

consideration. Moreover, the use of certain natural products may raise questions about possible allergic reactions in individuals.

Finally, the simplest, but also the most risky, method to lower TCA content in contaminated wine is to blend it with defect-free wine. This approach is not recommended and can only be accepted if the problematic wine has just a very minor TCA taint. The dilution can then reduce the TCA concentration below the sensory threshold levels. In other cases, there is a high risk that the entire volume of wine after blending will become defected.

In general, most of the methods described above are aimed primarily at large volumes of wine, while it is not yet bottled. Therefore, it is necessary to adapt these treatments to industrial scale processes, which may be less effective than test treatments on a laboratory scale. In addition, the cost efficiency of the presented treatments should be taken into account, as some of the methods can be relatively expensive.
